To date, vaccine adjuvants such as complete Freund's adjuvant derived from the cell wall of mycobacteria in a water-in-oil emulsion or aluminum salts have been developed using an empirical trial-and-error approach. This approach has identified compounds that compensate for poor immune responsiveness to an antigen, increase vaccine stability, and reduce the dose of an antigen required for protection, though the exact immunological mechanism for their function in the host is still under debate. However, identifying or developing new, rationally-designed adjuvant(s) that stimulate components of the host innate and/or adaptive immune systems, based on known correlates of immune protection are now possible with efficacy and safety being the primary goals. The significance of identifying novel adjuvants is highly important as most of the current trends in vaccine development are based on poorly immunogenic, highly purified antigens, which will require an appropriate adjuvant to induce an effective protective immunity.
An area of great interest is in the development of more rationally-designed adjuvants based on molecules that stimulate the host innate immune system, specifically pattern-recognition receptors, including toll-like receptors (TLR). The TLR family plays a critical role in early innate immunity by acting as sensors in response to invading pathogens and are expressed in tissues involved in immune function, e.g. peripheral blood leukocytes and spleen or those exposed to the external environment like the gastrointestinal tract and lung. To date, ten human and twelve murine TLRs have been identified with most localized to the cell plasma membrane with the exception of TLR3, TLR7, TLR8, and TLR9 being localized intracellularly. Each receptor recognizes highly conserved structural motifs expressed by microbial pathogens (PAMPs) that are different from host ligands. Briefly, TLR2 is essential for the recognition of bacterial lipoproteins, lipoarabinomannans and lipoteichoic acids from Gram-positive organism; TLR3 recognizes viral dsRNA; TLR5 detects flagellin, the major protein subunit of flagella; TLR9 recognizes hypo-methylated CpG DNA motifs; TLR7 and TLR8 recognize small synthetically derived viral RNAs; TLR4 is activated by LPS through its bioactive component lipid A or endotoxin, in conjunction with the accessory molecules, MD-2 and CD14 that form a complex with TLR4. Finally, TLR signaling specificities are extended by their ability to heterodimerize (they mostly homodimerize, but TLR2 heterodimerizes) with one another. Stimulation of TLRs by PAMPs initiates signaling cascades that involve a number of proteins, such as MyD88, TRIF and IRAK (Kawai and Akira, 2011; Pasare and Medzhitov, 2005). These signaling cascades lead to the activation of transcription factors, such as AP-1, NF-κB and IRFs inducing the secretion of pro-inflammatory cytokines, chemokines, and effector cytokines that direct or modify the host immune response. Among TLRs, only TLR3 and TLR4 stimulate the production of type I IFNs via TRIF and the induction of a robust IL-12p70 response that strongly enhances cellular-mediated and humoral immune responses.
Currently, a number of TLR mimetics are being used as stand-alone immunotherapeutic adjuvants or in combination with TLR signaling molecules. Examples include natural and synthetic-based lipid A mimetics, monophosphoryl lipid A (MPL) and aminoalkyl glucosaminide phosphates (AGPs). MPL is a chemically modified form of lipid A, derived from Salmonella minnesota R595 lipopolysaccharide. These chemical modifications result in the generation of a 4′-monophosphoryl, 3-O-deacylated lipid A structure that also displays differences in the overall number and location of individual fatty acids attached to the glucosamine sugar backbone of lipid A. As MPL is chemically derived, lot-to-lot differences in the microheterogeneity of the acyl groups make performing structure-activity relationship studies problematic. In contrast, the AGP classes of lipids are monosaccharide lipid A mimetics based on the biologically active hexa-acylated component present in MPL, chemically synthesized with modifications in the acyl chain length and location in uniform positions. Both molecules display low-toxicity, as compared to LPS (approximately 0.1% as toxic as LPS for MPL) and are potent immunostimulators of the host innate and adaptive immune system. Assessment of the adjuvant characteristics of MPL has shown it to be an effective adjuvant for the induction of both humoral and cell-mediated immunity in which MPL can induce both Th1- and Th2-type immune responses in the systemic and mucosal compartments of the immune system. MPL is currently a component in many of GalaxoSmithKline's (GSK) proprietary and novel adjuvant systems used in multiple GSK Bio vaccines. However, due to the increased heterogeneity from the chemical hydrolysis of lipid A for the production of MPL and the limitations and labor intense nature of synthesizing AGPs that more closely mimic the structure of naturally occurring lipid A structures, alternative technologies are in need.